The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds

Abstract

Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca-dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca-dependent Ser phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca ions [K(Ca) < 0.4 µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser, RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca-signaling and the control of photosynthate partitioning during COS development.

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds

“…S4) or a detergent such as 0.5% (v/v) Triton X-100 (results not shown). Similar results were reported for cytosolic CDPKs that, respectively, phosphorylate Suc synthase at Ser-11 in developing COS (Fedosejevs et al, 2016) and nitrate reductase in spinach leaves (Bachmann et al, 1996;Douglas et al, 1998).…”

“…It is notable that the developmental pattern of in vivo BTPC phosphorylation at Ser-451 in COS (Dalziel et al, 2012;Hill et al, 2014), as well as the basophilic phosphorylation motif flanking its Ser-451 residue (Supplemental Fig. S3), are distinct from those of COS PTPC and RcSUS1 (Tripodi et al, 2005;Fedosejevs et al, 2016). These differences imply specific protein kinasephosphatase pairings in controlling the phosphorylation status of BTPC, PTPC versus RcSUS1 in developing COS.…”

“…Furthermore, RcCDPK2 phosphorylates RcSUS1 at Ser-11 in developing COS and shows many additional and prominent differences compared to RcCDPK1 including: (i) a relatively nonspecific in vitro substrate selectivity, including abundant peptide kinase activity; (ii) a significantly lower K 0.5 (Ca 2+ ) value of approximately 200 nM; (iii) insensitivity to metabolite effectors including PEP; (iv) partial association with microsomal membranes; and (iv) a completely opposite developmental profile in COS (i.e. RcCDPK2 expression, RcSUS1 Ser-11 kinase activity, and in vivo RcSUS1 phosphorylation at Ser-11 progressively decrease during COS development; Fedosejevs et al, 2014Fedosejevs et al, , 2016.…”

“…1) that is typical of most CDPKs (Schulz et al, 2013). The closest paralog of RcCDPK1 is RcCDPK2 (70% sequence identity; Hill et al, 2014), the kinase that in vivo phosphorylates castor Suc synthase-1 (RcSUS1) at Ser-11 in developing COS (Fedosejevs et al, 2016).…”

“…Furthermore, all PTPCs and Suc synthases examined to date share an orthologous phosphorylation motif near their N-terminus, i.e. f -5 -X -4 -basic -3 -X -2 -X -1 -Ser-X +1 -X +2 -X +3 -f +4 (where f is a hydrophobic residue, X is any amino acid, and subscripts denote residue positions relative to the seryl phosphorylation site; Winter and Huber, 2000;O'Leary et al, 2011b;Fedosejevs et al, 2014Fedosejevs et al, , 2016. This differs from the unique and highly conserved BTPC recognition motif identified for RcCDPK1, namely: f -4 -X -3 -Basic -2 -X -1 -Ser-X +1 -X +2 -Basic +3 -f +4 (Dalziel et al, 2012;Hill et al, 2014;Supplemental Fig.…”